312 ISJ 14: 312-323, 2017 ISSN 1824-307X RESEARCH REPORT Evolution of the digestive enzymes and bacterial changes of the gastrointestinal tract of the Artemia urmiana during growth period H Ahmadniaye Motlagh 1 , O Safari 1 , M Farhangi 2 , M Lashkarizadeh-Bami 3 1 Department of Fisheries, Faculty of Natural Resources and Environment, Ferdowsi University of Mashhad, KhorasanRazavi, Iran 2 Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran 3 Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Accepted August 19, 2017 Abstract As the digestive enzymes and gastro-intestinal (GI) bacterial community contribute to the health and nutrition of the organism, this study aims to evaluate the ontogeny of bacterial population and digestive enzymes activities in the GI tract of Artemia urmiana from nauplii to the adult stage. Artemia cysts were hatched under standard conditions and stocked at a density of 20 nauplii ml -1 for 15 days. Samplings for growth, bacterial and enzymatic analysis were collected on days 1, 5, 10 and 15 of the experiment. The results indicated that the GI tract was sterile at the time of hatching. Artemia GI tract was active after hatching by protease (0.282 ± 0.001 U mg -1 protein min -1 ), lipase (0.182 ± 0.001 U mg - 1 protein min -1 ) and amylase (0.295 ± 0.001 U mg -1 protein min -1 ) secretion and increased during the experiment. In addition, a significant relation (p < 0.05) was observed between Artemia total length and the activity of digestive enzymes (lipase (r 2 = 0.98) amylase (r 2 = 0.97) and protease (r 2 = 0.98). A significant relation (p < 0.05) was observed between total aerobic bacteria and digestive enzyme secretion (lipase, r 2 = 0.83; amylase, r 2 = 0.66 and protease, r 2 = 0.84) too. Such a relation was observed between total Bacillus spp. count and digestive enzyme activities (lipase r 2 = 0.82; amylase r 2 = 0.79; and protease; r 2 = 0.74). These results suggest that in addition to the chemical composition of food, total length, GI bacteria enzyme secretion and the interaction of these factors contribute in digestive enzymes ontogeny during the growth period. Key Words: Artemia; health; nutrition; digestion Introduction Some aquatic organisms, consume bacteria as food and gastrointestinal (GI) flora can be a reflection of bacterial community swallowed with food or as food. Bacteria population can play an important role as food by providing micronutrients such as essential fatty acids, vitamins, or digestive enzymes (Olafsen, 2001). The GI flora is affected by several factors such as season (Irianto and Austin, 2002), species (Cahill, 1990), diet, environmental conditions and the growth stage (Hansen and Olafsen, 1999). The microbial ecology of the GI tract of a variety of freshwater and marine organisms has been investigated (Denev et al., 2009; Zarkasi et al., 2016; ___________________________________________________________________________ Corresponding author: Hamidreza Ahmadniaye Motlagh Department of Fisheries Faculty of Natural Resources and Environment Ferdowsi University of Mashhad KhorasanRazavi, Iran E-mail: ahmadnia@um.ac.ir Zhu et al., 2016). In addition, some researchers have studied the probiotic properties of some indigenous and non-indigenous bacteria in aquaculture, but information on Artemia larval development (ontogeny) is scarce and no work has been published concerning the bacterial changes of Artemia gut during development. Investigations of nutritional requirements and feeding ecology of marine invertebrates requires a deep consideration of basic digestive physiology. (Patel et al., 2012). Structure and the physiology of the digestive system of crustaceans and other marine invertebrate undergo important ontogenetic changes with adaptation to different food types (Andrés et al., 2010) and developmental stages (Veróonica and Gimenez, 2013). Fulfilment of nutritional requirements of marine invertebrates, including crustaceans, plays a critical role during larval development. Since digestion is considered as a key factor in metabolism, it may be essential in determining the availability of nutrients required for biological functions (McConaugha, 1985). Thus, the mailto:ahmadnia@um.ac.ir 313 Table 1 Physico chemical parameters of Artemia hatching and rearing water (mean ± SD) pH Salinity(gL -1 ) DO (mgL -1 ) Temperature (ºC) Agent 8.3±0.5 35±2 4±1 29±1 Hatching 8.3±0.5 60±2 6±1 29±1 Rearing study of various aspects of the digestion physiology during ontogeny is essential to have a promising larviculture (Applebaum et al., 2001). Most of the reports on digestive enzymes in crustaceans have been confined to adult specimens; interactions between variations in gut morphology, digestive enzyme activities, and diet throughout early stages of life cycle are thoroughly unknown. Changes in digestive enzyme activity during development have been reported in relatively few crustacean species Penaeus setiferus (Lovett and Felder, 1990) Artemia spp, (Gawlicka et al., 2000), Macrobrachium rosenbergii (Kamarudin et al., 2011), Litopenaeus vannamei (Wei et al., 2014). Despite digestive enzymes secreted by indigenous bacteria of GI are of high importance (Suzer et al., 2008; Wang et al., 2017), we found no study concerning the role of bacteria in enzymatic development of crustaceans. Ceccaldi (1989) stated that digestive enzymes of crustacean species are diversified into proteases, among which trypsin is the major one, lipases and esterases, amylases, maltases and chitinases are also well-represented. Determination of digestive enzyme profiles in puerulus, postpuerulus, juvenile and adult stages of the spiny lobster Jasusedwardsii was made to find out the ontogenetic alterations in digestive proficiencies. A variety of enzymes was observed in juvenile and adult lobsters, exhibiting their potential to exploit different diets. According to the results, lobster was found out to be carnivorous (Johnston, 2003) as dietary protein played more essential role than carbohydrate sources. Gawlicka and co-workers in 2000 determined Activities of trypsin, amylase, lipase and alkaline phosphatase in metamorphic larvae Hippoglossus hippoglossus and in their Artemia prey to estimate the importance of exogenous enzymes for Atlantic halibut larvae. The calculated contribution of enzyme activities derived from Artemia prey to the relatively high levels of enzyme activity in the digestive system of metamorphic larvae was less than 10 % for all enzymes except amylase, for which the contribution was estimated to be more than 50 %. Based on the available literature, there is no information on GI bacterial ontogeny and the interaction between GI bacteria and digestive enzymes, this survey can open new viewpoint of the nutritional biology of Artemia and other crustaceans. Recently, Artemia urmiana (Günther, 1890) population as an indigenous species in Urmia Lake (in the North-west of Iran) has been exposed to main challenges including drought and salinity increment (Motlagh et al., 2012). Therefore, indoor mass-culture of Artemia can alleviate the pressure of aquaculture industry on natural stocks to collect following products (cyst and biomass). Regarding the critical role of GI microflora and digestive enzymes on the health and nutrition of aquatic larvae, the aim of present study was to evaluate the digestive enzymes (protease, lipase and amylase) and bacterial ontogeny of the GI tract of the A. urmiana from nauplius to the adult stage as a vital point of Artemia mass culture. Materials and Methods Hatching and rearing of Artemia urmiana Nauplii of A. urmiana were hatched in the laboratory in cylindrical containers (2 L) of saltwater and maintained on a 12:12 light:dark cycle, under standard condition (Sorgeloos 1986). Artemia nauplii were transferred into 60 L polyethylene tanks with the density of 20 nauplii ml -1 and reared for 15 days (Shahnaz Jabari et al., 2015). Physicochemical properties of hatching and rearing water, including temperature, dissolved oxygen, salinity and pH (Table 1) were monitored daily according to standard methods (Agh, 2007) for hatching and rearing periods. Artemia urmiana feeding During the first five experimental days, nauplii were fed with backers yeast (Saccharomyces cerevisiae) (Lavens and Sorgeloos, 1996). From the second day, after hatching 1.25 mg of baker yeast per 1000 nauplii in 400 ml saline water (35 gL -1 ) at 28 ºC, the distribution of solutions in rearing water was facilitated by passing them through a 150 micron mesh. From the sixth day onwards, the Artemia were fed a diet including white wheat flour (11.24 %) and equal amounts of soybean meal and chickpea flour (44.38 %) provided by Behparvar Co. (Iran). Feeding was performed three times a day with a four-hour interval. The dietary chemical composition was analyzed using the standard methods (Peterson and Martin-Robichaud, 1983). The crude protein, crude fat, dry matter and ash values were (12 % ± 0.93), (55 % ± 1.2), (97.5 % ± 0.77) and (5 % ± 0.35), respectively. Table 2 shows the feeding schedule of Artemia. Growth monitoring Biometry of Artemia was performed on the first, fifth, tenth and fifteenth experimental days for the evaluation of growth. Sampling was conducted by removing five samples of water with 10 ml volumes from each tank and measuring the total length of Artemia by a micrometer (0.01 mm). 313 Table 2 Artemia feeding schedule over the experiment 11-14 10 9 8 7 6 5 4 3 2 1 Experimental days 0.07 - 0.07 0.062 0.06 0.05 - 0.05 0.03 0.02 - Feed amount (g L-1) - - - 0.0155 0.03 0.037 - 0.05 0.03 0.02 - Yeast 0.07 - 0.07 0.0465 0.03 0.0125 - - - - - Dry food Microbial analysis The total aerobic bacteria count and total Bacillus count in the Gl of Artemia (CFU g -1 Artemia) were estimated on first, fifth, tenth and fifteenth days. A 70 % alcohol was used for washing nauplii, and the sterile saline water (35 g NaCl L-1) was used for rinsing them to eliminate the sticking bacteria from their body surface. Five ml of sterile saline water was gradually added to the samples in order to homogenize them. Then, ten times serial dilution were prepared, and mediums Bacillus cereus Agar and Marine agar 2216 (HiMedia©) were used to count the total aerobic bacteria and Bacillus, respectively (Rengpipat et al., 1998). Bacillus cereus Agar was incubated at 30 °C for 24 h, and Marine agar was incubated at 29 °C for 24 h. Enzymatic assays Samplings for determining the activities of protease, lipase and amylase were conducted on first, fifth, tenth and fifteenth experimental days. The samples were first washed with cold fresh water and rinsed. Then, the samples were stored in 15-ml falcon tubes and instantly transferred to a freezer (- 80 ºC) (Paiva-Maia et al., 2013). The samples were defrosted in laboratory conditions to extract the enzymes. The homogenates of the whole animal body were used in all evaluations. Homogenization of the extracts prepared in physiological saline solution (0.9 % NaCl) was performed by adding saline solution which obtained a total volume of 1.6 ml per sample. The homogenized solutions were centrifuged at 5,000g for 5 min. The enzymatic evaluations were conducted using the supernatants. The hydrolysis of casein at pH 8 was used to estimate the protease activity (Paiva-Maia et al., 2013), starch was used as the substrate to measure the amylase activity (Worthington, 1991). In order to determine the lipase activity, olive oil emulsion substrate-Gum Arabic was used and the thawed samples were titrated at room temperature (Worthington, 1991). Statistical analysis All percentage data were transformed using the arcsine method. The Leaven test was used to confirm the homogeneity of variance, and the Kolmogorov-Smirnov test was used to determine the normality of data (Zar, 1999). The data were analyzed using one-way ANOVA. Duncan multiple range test was applied in order to find out if there were any significant differences among the treatments (p < 0.05). Regression relations were conducted using SPSSTM version 19 (SPSS Inc., Chicago, USA). Results The changes of total length in Artemia urmiana As shown in Figure 1, there are significant differences between total length of A. urmiana during sampling days. The significantly highest total length (3.5 mm) was observed in the day 15. Fig. 1 The mean (±SD) total length (mm) in A. urmiana during sampling times with three replicates. Different letters indicate significant differences (p < 0.05). 314 313 Fig. 2 The mean (±SD) total aerobic bacterial count (×10 6 CFU g -1 ) in A. urmiana GI during sampling times with three replicates. Different letters indicate significant differences (p < 0.05). The ontogeny of the GI bacterial in Artemia urmiana The changes of the total aerobic bacterial count Total number of aerobic bacteria in the GI of newly hatched Artemia nauplii showed that the GI was sterile and free of bacteria. As shown in Figure 2, the total number of aerobic bacteria shows a significantly (p < 0.05) increasing trend, up to the fifteenth day of the experiment. The average number of total aerobic bacteria in the gut showed significant differences between the fifth, tenth and fifteenth day of the experiment (p < 0.05). The results revealed no significant difference between the tenth and the fifteenth day. The changes of the Bacillus spp. count No Bacillus was reported in Artemia GI at the first day of sampling but count increased later throughout the experiment. The final comparison between average Bacillus spp. count shows a significant increase (p < 0.05) from zero (in day one) to 3.8 ± 0.8×104 (in day 15) (Fig. 3). The ontogeny of digestive enzymes activities in Artemia urmiana The changes in protease, amylase and lipase activities during the experiment are shown in Figure 4. On the first day, the specific activities (U min -1 ) of protease, amylase and lipase in the Artemia gut were reported to be 0.282 ± 0.07, 0.295 ± 0.05 and 0.182 ± 0.02, respectively. The lipase activity was higher than the other enzyme activities (p < 0.05) on day five. However, on the tenth and fifteenth days, significant differences were observed among all enzymes, and lipase exhibited the highest activity (p < 0.05). At the beginning of the experiment, amylase showed the highest activity with no significant difference with the others, however over the time amylase activity decreased and lipase activity was increased. Fig. 3 The mean (±SD) Bacillus spp. count (×10 6 CFU g -1 ) in A. urmiana during sampling times with three replicates. Different letters indicate significant differences (p < 0.05). 315 313 Fig. 4 The mean (± SD) activities (U mg protein -1 min -1 ) of digestive enzymes (lipase, amylase and protease) in A. urmiana during sampling times with three replicates. Different letters indicate significant differences (p < 0.05). Small letters (a - d) and capital letters (A - C) show differences during day post hatch and among enzyme activities during a specific day, respectively. Polynomial models in Figure 5 indicated that, there were significant difference (p < 0.05) between polynomial models of length and digestive enzyme activities including lipase (r 2 = 0.98), amylase (r 2 = 0.97) and protease (r 2 = 0.98). As well, Figure 6 significant differences were found (p < 0.05) between polynomial models of the age of Artemia (Day Post Hatch) and digestive enzymes including lipase activity (r 2 = 0.99), amylase activity (r 2 = 0.99) and protease activity (r 2 = 0.99). Significant differences were found (p < 0.05) between polynomial models of total aerobic bacteria count and digestive enzymes including lipase activity (r 2 = 0.83), amylase activity (r 2 = 0.66) and protease activity (r 2 = 0.84) (Fig. 7). In addition, as shown in Figure 8, there were significant differences between (p < 0.05) polynomial models of total Bacillus spp. count and digestive enzymes including activities of lipase, amylase and protease (r 2 = 0.82, 0.79 and 0.74, respectively). Discussion The ontogeny of the GI bacteria in Artemia urmiana Ontogeny of the bacterial flora of the GI tract of freshwater and marine organisms have not been studied yet and this issue requires more research (Safari et al., 2014; Wang et al., 2017). In the current study, no bacteria were detected in GI on the first day post-hatch which confirms the other researchers finding (Ringø and Gatesoupe, 1998). An increasing trend of total aerobic bacteria was observed. In fact, the total number of aerobic bacteria increased significantly with Artemia growth and development of the attachment sites in the GI. This trend was also observed in total Bacillus count as representative beneficial bacteria. This could be due to dietary consumption. In the present study, the Artemia consumed S. cerevisiae for the first 5 days of the experiment. Several researchers had proved the dietary effects on the intestinal microflora. The effect of the food sequence on the population of intestinal microbiota in larvae and juveniles of Sparus aurata has been observed by weaning the early juveniles of this fish (Savas et al., 2005). Vibrio and Pseudomonas were found out as the dominant genera after feeding juveniles with a compound diet depending on culture methods (Silva et al., 2011). Unfortunately, few studies have been conducted on Artemia ontogeny, which requires further research. Moreover, quantifying the main bacteria family throughout each ontogeny stage and using DGGE technique are recommended as the future research topics. The ontogeny of digestive enzyme activities in Artemia urmiana One of the most important factors affecting commercially successful larval production is survival rate that is affected by various factors-mainly, larval rearing period and larval nutrition (Shields, 2001; Turkmen et al., 2017). Digestion of food to provide nutrients for growth, maintenance, motion, and reproduction is a very important function in an organism (Hernández and Murueta, 2009). The intake of sufficient amount of nutrients and their efficient digestion by an organism depends on its ability to select food and the capacity of its digestive enzymes (Bautista 1983; Silva et al., 2010). It is thus important to understand the profile of these digestive enzymes, as this can be used as a tool to develop food specific to the metabolic needs of species (Pavasovic et al., 2004). However, we need to standardize specific histochemistry methods for further studies on live feed production industry including Artemia. Extended published records have focused on invertebrate digestive enzyme synthesis, regulation, and evaluation, including crustaceans (Le Vay et al., 2001; Muhlia-Almazán et al., 2008; Andrés et al., 2010; 316 313 Fig. 5 Polynomial model fitting (a) lipase activity (U/mg protein. min -1 ), (b) amylase activity (U/mg protein. min -1 ) and (c) protease activity (U/mg protein. min -1 ) to length (L; mm) in A. urmiana with three replicates. a) b) c) 317 313 Fig. 6 Polynomial model fitting (a) lipase activity (U/mg protein. min -1 ), (b) amylase activity (U/mg protein. min -1 ) and (c) protease activity (U/mg protein. min -1 ) to day post hatch (D) in A. urmiana with three replicates. c) b) a) 318 313 Fig. 7 Polynomial model fitting (a) lipase activity (U/mg protein. min -1 ), (b) amylase activity (U/mg protein. min -1 ) and (c) protease activity (U/mg protein. min -1 ) to total count (TC; x 10 6 CFU g -1 ) in A. urmiana with three replicates. a) b) c) 319 313 Fig. 8 Polynomial model fitting (a) lipase activity (U/mg protein. min -1 ), (b) amylase activity (U/mg protein. min -1 ) and (c) protease activity (U/mg protein. min -1 ) to Bacilus spp. count (BC; x 10 4 CFU g -1 ) in A. urmiana with three replicates. c) b) a) 320 313 Veróonica and Gimenez, 2013; Wei et al., 2014). Assessment of the digestive enzymes activity can be used as an indicator of larval growth rate, food acceptance and the digestive capacity. Several workers have reported the activity of proteases, amylases, and lipases in crustaceans (Figueiredo et al., 2001). But no report was found about A. urmiana digestive enzymes (protease, lipase and amylase) ontogeny during development. It has shown that many crustacean species with economic importance possess the necessary enzymes for the hydrolysis of carbohydrates; however, amylase has been poorly studied in crustaceans (Pavasovic et al., 2004). The results of the current experiment showed that A. urmiana nauplii digestive system is active starting from hatching in production and secretion of digestive enzymes. These findings were in agreement with Andrés et al. (2010) which indicated that all assayed enzymes were active from hatching time in spider crab, while Lovett and Felder (1990) reported that in Penaeus setiferus no activities were present for pepsin and lipase in some developmental stages. Maximum activities of protease, lipase and amylase were recorded in the fifteenth day of the experiment for A. urmiana which agrees with peak activities for all enzymes occurred during late zoea or early mysis larval stages of P. setiferus. This discrepancy can be species-specific, due to sampling methods and the definition of critical ranges of digestive enzyme activities during sample preparation and final optical density (OD) reading. The activity of lipase was higher than others during the test that is consistent with other studies (Johnston, 2003). The high lipase activity in all Artemia stages reflects the importance of lipid as an energy reserve in crustaceans (Icely and Nott 1992; Johnston, 2003) and its key role as an energy substrate for J. edwardsii puerulus (Jeffs et al., 1999, 2001, 2002), H. americanus (Sasaki et al., 1986) and Artemia. According to Johnston (2003), a significant change in lipase-specific activity during different post hatch days confirms that lipid utilization is not consistent throughout development. Although the lipase activity was higher than other assessed enzymes, such an increment will not necessarily demonstrate higher absorption of fat in the digestive tract. Results indicated that by shifting diet to a rich protein diet on the fifth day, the secretion of protease increased significantly. These findings are confirmed by other researchers who treated A. urmiana with different diets and reported that chemical composition of diets can alter the activities of digestive enzyme secreted by Artemia (Bami et al., 2011). Similar results were obtained in the investigation of the ontogenetic changes in digestive enzymatic capacities of the spider crab (Maja brachydactyla) (Andrés et al., 2010). On the other hand, some hypothesis on considering diet as a non-essential factor for ontogenic changes in the activities of digestive enzymes may arise. Instead, some developmental changes in enzyme synthesis or a secondary effect of change in the function and approximate size of the midgut throughout its distinction may be responsible for the ontogenic alteration of digestive enzyme activities (Lovett and Felder, 1990). Results reported there was a significant relationship between Artemia total length and the protease activity which it may suggest that larger Artemia tends to consume foods that contain more protein content, like certain protozoa and bacteria. A similar trend was detected in ontogenetic changes in digestive enzyme activity of the spiny lobster (Jasus edwardsii) that revealed a strong relationship between total enzymes activity and carapace length juvenile and adult lobsters (Johnston, 2003). In the present study, we observed significant polynomial models between total length and digestive enzymes activities. These findings are in agreement with Ribeiro and Jones (2000) which stated that the evolution of trypsin and lipase activity in Fenneropenaeus indicus showed a dependence on the length of the post larvae, therefore the enzymatic response increases with size and evolutionary stages in F. indicus. Measurement of amylase and trypsin activities during different evolution stages of San Francisco Lake Artemia showed that the secretion of these enzymes depends on nutritional requirements of Artemia, and also the amount, chemical composition and the ingestion of food (Harris et al., 1986). These researchers also have reported lots of changes in the activity of these enzymes in different developmental stages. These enzymes activity can fluctuate depending on the age and type of food. Since the beneficial bacteria of GI tract like Bacillus spp. are capable of secreting digestive enzymes (Moriarty, 1998), it is likely that a part of the enzyme activity in the GI was associated with the bacteria. These suggest that in addition to chemical composition of food (Bami et al., 2011), total length, evolutionary stages, GI bacteria enzyme secretion (Motlagh et al., 2012), the interaction among these factors (Augusto E et al., 2012) can influence on digestive enzymes activity during growth period. However, these findings may open a new area in live feed production industry. The results of this study demonstrated the role of GI bacteria and other factors in the secretion of digestive enzymes. The accurate discovery of the bacterial role in producing digestive enzymes may result in the selection of probiotics more compatible with crustaceans and more accurate application of prebiotics. However, it requires a precise identification of bacterial communities and their role in the production and secretion of each digestive enzyme. Nowadays, new methods such as NGS and DGGE have been introduced to obtain such an intention. 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